Tissue maceration or fragmentation

The capacity of young fern tissue to regenerate adventitious shoots can be very high. Fern prothallus tissue (the gametophyte generation produced from germinating spores) has a high capacity for regeneration; a new prothallus can usually be grown from small isolated pieces of tissue (Whittier and Steeves, 1962), or even from single cells produced by maceration (Miller J.H., 1968; De Fossard, 1976; Knauss, 1976). Plants can also be regenerated from homogenised sporophyte tissue of some fern genera, and homogenisation has been incorporated into tissue culture, or partial tissue culture techniques for the propagation of plants of this class (see Volume 2).

Because a high proportion of the direct cost of micropropagation is attributable to the manual separation and transfer of explants and cultured material between media, the ability to regenerate plants from macerated or fragmented tissue would be extremely advantageous. Unfortunately there seem to be only a limited number of publications describing the formation of shoots directly from machine-macerated tissue of higher plants. One of them is the patent of Lindemann (1984), the claims of which may have been somewhat optimistic. Also Levin et al. (1997) reported on the regeneration of different plant species using a homogenisation technology. However, shoot regeneration from fragmented shoot tips, or micropropagation of some plants by culturing shoot material or tissue fragments in fermentors (Vol. 2), are somewhat comparable.

3.2.2. Organised calluses

In most callus cultures, shoots are produced from meristems which arise irregularly and may therefore be genetically altered. By contrast, so-called 'organised' or 'semi-organised' calluses are occasionally isolated in which there is a superficial layer of proliferating shoot meristems, overlaying an inner core of vacuolated cells acting as a mechanical and nutritional support. Calluses of this kind were termed organoid colonies by Hunault (1979): the names meristemoids and nodules have also been proposed. A meristemoid is defined as a cluster of isodiametric cells within a meristem or cultured tissue, with the potential for developmental (totipotential) growth. Meristemoids may give rise to plant organs (shoots, roots) or entire plants in culture (Donnelly and Vidaver, 1988). Nodules also comprise meristematic cells, but they are distinct from meristemoids because they are independent spherical, dense cell clusters which form cohesive units, with analogy to both mineral nodules in geology and root nodules of legumes (McCown et al., 1988). Nodule culture has been extensively used for the propagation of Cichorium intybus (Pieron et al., 1993).

The presence, in meristemoids, of an outer layer of shoot meristems seems to inhibit the unbridled proliferation of the unorganised central tissue (Hussey, 1983). Geier (1988) has suggested that the control mechanisms which ensure the genetic stability of shoot meristems are still fully, or partly, active. Maintenance of a semi-organised tissue system depends on a suitable method of subculture and upon the use of growth regulator levels which do not promote excessive unorganised cell growth. Repeated selective transfer of unorganised portions of an organised Anthurium scherzerianum callus eventually resulted in the loss of caulogenesis (Geier, 1986). Conversely, by consistently removing the unorganised tissue when subculturing took place, shoot formation from the callus was increased.

Cultures consisting of superficial shoot meristems above a basal callus, seem to occur with high frequency amongst those initiated from meristem tip, or shoot tip, explants. Hackett and Anderson (1967) induced the formation of tissue of this type from carnation shoot tips by mutilating them with a razor blade before culture. Similar cultures were also obtained from seedling plumular tip explants of two (out of five tested) varieties of Pisum sativum placed on an agar medium (Hussey and Gunn, 1983; 1984). The calli were highly regenerative for 2-3 years by regular subculture to agar or shaken liquid medium. Maintenance was best achieved with an inoculum prepared by removing larger shoots and chopping the remainder of the callus and small shoots into a slurry. A callus, formed at the base of Solanum curtilobum meristem tips on filter paper bridges, gave rise to multiple adventitious shoots from its surface when transferred to shake culture in a liquid medium (Grout et al., 1977).

Callus with superficial proliferative meristems has also been induced by culture of shoot or meristem tips on a rotated liquid medium, in:

• Nicotiana rustica (Walkey and Woolfitt, 1968);

• Chrysanthemum morifolium (Earle and Langhans 1974c);

In Stevia rebaudiana (above), a slow rotation speed (2 rpm) was essential for initiation of an organised callus. A small callus formed upon the explant and in 2-3 weeks came to possess primary superficial shoot primordia which were globular and light green. Dark green aggregates of shoot primordia (termed 'secondary shoot primordia'' by Miyagawa et al., 1986) were developed within 6 weeks. If divided, the aggregations of shoot initials in both Nicotiana and Stevia could be increased by subculture or, if treated to a different cultural regime, could be made to develop into shoots with roots. Shoots were produced from Chrysanthemum callus upon subculture to an agar medium. A spherical green dome-like structure was produced from meristem tips dissected from germinated Eleusine coracana (Gramineae) caryopses. When cut into four and subcultured, a green nodular structure was formed which grew to 5-10 mm in diameter. It was similar in appearance to a shoot dome, but much larger (a natural shoot dome is only 70-80 ^m wide). The nodular structures were termed 'supradomes' by Wazizuka and Yamaguchi (1987) because, unlike normal callus, superficial cells were arranged in an anticlinal plane and those beneath had a periclinal arrangement. Numerous multiple buds could be induced to form when the organised tissue was subcultured to a less complex medium.

Although proliferative meristematic tissue formed from shoot tips always appears to be accompanied by a basal callus, the superficial meristematic cells may well be derived directly from the cells of the apical shoot meristem of the explant, for they preserve the same commitment to immediate shoot formation. The presence of the apical meristem in the explant seems to be essential and culture of tissue immediately beneath it does not produce a callus with the same characteristics (Hussey and Gunn, 1984). Similar semi-organised callus can appear at the base of conventional shoot cultures. In the green granular callus mass which formed at the base of Rhododendron shoot tips, each granule represented a potential shoot (Kyte and Briggs, 1979). Organised caulogenic callus is thus closely comparable to embryogenic callus formed from pre-embryogenically determined cells.

Organised callus can be produced from explants other than shoot tips; in Anthurium, it has been derived from young leaf tissue (above, Geier, 1986) and from spadix pieces (Geier, 1987).Organised callus has two characteristics which distinguish it from normal unorganised callus: the plants produced from it show very little genetic variation, and it can be subcultured for a very long period without losing its regenerative capacity. The callus of Nicotiana (above) was able to produce plantlets over a ten-year period, while that of Chrysanthemum gave rise to plants continuously during four years.

The use of cultures with superficial proliferative meristems has not yet been widely used for micropropagation. There are three possible reasons:

• the genetic variation which is almost invariably induced by shoot regeneration from normal callus, has cautioned against the use of any sort of callus culture for this purpose;

• organised callus may not always be readily distinguished from its unorganised counterpart;

• methods of initiating organised callus in a predictable fashion have not yet been fully elucidated.

There are examples of the initiation of organised callus from a sufficiently wide range of plant species (particularly from meristem tip explants) to suggest that it could be a method of general applicability. Multiplication may well be amenable to large-scale culture in fermentors (Vol. 2).

3.2.3 Direct embryogenesis

Somatic embryos are often initiated directly upon explanted tissues. Of the occurrences mentioned in Chapter 1, one of the most common is during the in vitro culture of explants associated with, or immediately derived from, the female gametophyte. The tendency for these tissues to give rise to adventitious somatic embryos is especially high in plants where sporophytic polyembryony occurs naturally, for example, some varieties of Citrus and other closely related genera.

Ovules, nucellar embryos, nucellus tissues and other somatic embryos are particularly liable to display direct embryogenesis. In Carica somatic embryos originated from the inner integument of ovules (Litz and Conover, 1981a,b) and in carrot tissue of the mericarp seed coat can give rise to somatic embryos directly (Smith and Krikorian, 1988).

The nucellus tissue of many plants has the capacity for direct embryogenesis in vitro (Haccius and Hausner, 1976; Eichholtz et al., 1979; Rangaswamy, 1982; Litz, 1987). As explained in Chapter 1, explants may also give rise to a proliferative tissue capable of embryogenesis. The high embryogenic competence of the nucellus is usually retained during subsequent cell generations in vitro, should the tissue be induced to form 'callus' (or cell suspensions). It is not clear whether all cells of the nucellus are embryogenically committed. In Citrus, somatic embryos are formed from the nucellus even in cultivars that are normally monoembryonic (i.e. the seeds contain just one embryo derived from the zygote), whether the ovules have been fertilised or not. It has been suggested that only those cells destined to become zygotic proembryos can become somatic proembryos or give rise to embryogenic callus (Sabharwal, 1963); somatic embryos have been shown to arise particularly from the micropylar end of Citrus nucellus.

Adventitious (adventive) embryos are commonly formed in vitro directly upon the zygotic embryos of monocotyledons, dicotyledons and gymnosperms, upon parts of young seedlings (especially hypocotyls and cotyledons) and upon somatic embryos at various stages of development (especially if their growth has been arrested). The stage of growth at which zygotic embryos may undergo adventive embryogenesis is species-dependent: in many plants it is only immature zygotic embryos which have this capacity. Unfortunately, as the phenotypic potential of seedlings is rarely known, using them as a source of clonal material is of limited value.

Embryogenic determination can be retained through a phase of protoplast culture. Protoplasts isolated from embryogenic suspensions, may give rise to somatic embryos directly, without any intervening callus phase (Miura and Tabata, 1986; Sim et al., 1988). Treating protoplasts derived from leaf tissue of Medicago sativa with an electric field, induced them to produce somatic embryos directly upon culture (Dijak et al., 1986).

Adventitious embryos arising on seedlings are sometimes produced from single epidermal cells (Konar et al., 1972a; Thomas et al., 1976 - see Chapter 1). Zee and Wu (1979) described the formation of proembryoids within petiole tissue of Chinese celery seedlings, and Zee et al. (1979) showed that they arose from cortical cells adjacent to the vascular bundles which first became meristematic. Hypocotyl explants from seedlings of the leguminous tree Albizia lebbek showed signs of cracking after two weeks of culture and frequently young embryoids emerged (Gharyal and Maheshwari, 1981). Stamp and Henshaw (1982) found that primary and secondary embryogenesis occurred in morphogenically active ridges produced on the surface of cotyledon pieces taken from mature cassava seeds.

Somatic embryos have been observed on the roots and shoots of Hosta cultures (Zilis and Zwagerman, 1980) and on the needles and cultured shoots of various gymnosperm trees (Bonga, 1976; McCown and Amos, 1982).

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